Every year in the United States, more than 12,000 people suffer debilitating spinal cord injuries (SCI) - the result of numerous causes including accidents involving motor vehicle, sports, work, etc. Although investigation of SCI has progressed, an effective therapy to restore function remains the goal. Examining factors involved in central nervous system (CNS) cell death, fiber damage, neuron protection and neural tissue stability over time is important to the genesis of potent injury treatments. While in SCI, damaged neurons die of necrosis, a significant number of neurons in the penumbra later die of apoptosis or programmed cell death. Since calpain has been previously implicated as crucial in tissue destruction and cell damage following SCI, the long-term objective of this study is to investigate its role in the cell death mechanism in the lesion and potentially salvageable penumbra following injury and develop means for cell rescue and/or repair with preservation or restoration of function. Our preliminary results have shown mitochondrial damage, increased intracellular Ca2+ and calpain activity, increase pro-apoptotic mediators (e.g., cytochrome c release) and neuronal cell death in the lesion and penumbra following injury. From these findings, we propose the following broad spectrum hypothesis: In SCI, increased intracellular Ca2+ overload will cause calpain activation and loss of calpastatin regulation, leading to cell death while Ca2+ will damage mitochondria, increase mitochondrial permeability transition pore, induce cytochrome c release for caspase activation and cell death. Both calpain and caspase inhibitors, together with other agents, may protect neurons and preserve function. The following specific aim outline the investigational approach: (1) Investigate the contributions that increased intracellular calcium and calpain, los of calpastatin regulation to neuronal death and mitochondrial damage in SCI; (2) Study the role of pro-apoptotic factors, Bax/Bcl-2, cytochrome c and caspase-3 in mediating neuron and glial cell dysfunction/death in SCI; (3) Examine the therapeutic effect of calpain/caspase inhibitors, together or in combination with agents, affecting other destructive pathways in S( and assess cellular protection and function; (4) Study the role of oxidative stress and glutamate toxicity in primary culture cortical neurons, motor neuron cell line and glial cells and determine cell function before and after treatment with calpain/ caspase inhibitors and how this relates to events in trauma. Studies will use relevant and novel techniques to determine Ca2+ levels in cultured cells and SCI tissue slices, cell death by combined TUNEL/immunofluorescent labeling and cell function by electrophysiology. Understanding the mechanisms c cell death and cell protection in relation to calpain/caspase will establish inhibition of protease(s) as a viable SCI treatment.